Monosaccharide-Based Synthetic TLR4 Agonist Enhances Vaccine Efficacy against Pseudomonas aeruginosa Challenge

Abstract

Vaccine adjuvants are critical to improve the immunogenicity, efficacy, and durability of vaccines; however, their development has lagged behind that of vaccine antigens. Monophosphoryl lipid A (MPLA), a clinically approved adjuvant that stimulates Toll-like receptor 4 (TLR4), faces manufacturing challenges due to its complex and long synthesis. With the aim of simplifying the structure of MPLA while retaining its biological activity, we developed monosaccharide-based molecules FP18 and FP20Rha that activate TLR4 signaling. Both TLR4 agonists induced robust antibody activity against the model antigen, ovalbumin. Here, we report the potential of these TLR4 agonists to enhance the protective efficacy of the well-characterized OprF antigen against P. aeruginosa infection. OprF adjuvanted with FP18 showed reduced bacterial loads in lungs and spleens, relative to antigen alone in an acute P. aeruginosa pneumonia model. FP18-adjuvanted OprF also enhanced the production of anti-OprF antibodies and stimulated IFNγ and TNF in CD4⁺ T cells, suggesting a Th1-skewed cellular immune response. These adjuvants have promise for accelerating the development of effective vaccines against P. aeruginosa and other infectious diseases.

Keywords: P. aeruginosa; TLR4 agonist; acute pneumonia; infectious diseases; monosaccharide-based adjuvant; vaccine adjuvants; vaccines.

Figure 1

Chemical structure of monophosphoryl lipid A (MPLA) and the FP compounds used in the study: FP18 and FP20Rha.

Figure 2

Bacterial lung colonization and dissemination to spleens. (A) Timeline of immunization (50 μg rOprF alone; 50 μg rOprF plus 25 μg FP18; 50 μg rOprF plus 25 μg FP20Rha or 50 μg rOprF plus SAS (∼25 μg of active component) in 100 μL), blood collection, and P. aeruginosa challenge. (B,C) Bacterial clearance after immunization with rOprF adjuvanted with FP18 and FP20Rha or SAS adjuvant. Immunization of OprF in combination with SAS and FP18 significantly decreased P. aeruginosa colonization in the lungs by 1.5 log and 1.15 log, respectively (Kruskal–Wallis test, p = 0.003 and p = 0.026). rOprF + FP18 significantly decreased bacteria dissemination to the spleens relative to the immunization with antigen alone (Kruskal–Wallis test, p = 0.003). Each point represents a single mouse (n = 7) and the mean ± SD.

Figure 3

Seroconversion in response to adjuvanted OprF at 35 days post-immunization. Determined by ELISA of rOprF-specific IgGs in sera collected from immunized groups: rOprF (black circles), OprF + SAS (green squares), rOprF + FP18 (red triangles) and rOprF + FP20Rha (blue triangles). Values represent mean OD ± SD (A,C,E) or AUC ± SD (B,D,F). The sera of the seven mice were pooled and technical triplicates were performed (A,C,E). The AUC of each technical triplicate was calculated and is represented as a single point in the graphs (B,D F).

Figure 4

(A) Schematic illustration of the experimental timeline of the mouse study. Spleens were aseptically collected, disrupted, and filtered to obtain single-cell suspensions. One million splenocytes per well (in triplicate) were cultured with 10 μg/mL OprF for 60 h in RPMI supplemented with 10% FBS and 1% penicillin–streptomycin. (B) Percentages of singlet cells expressing the CD3 marker. (C) Percentages of parent cells expressing the CD4 marker. (D) Percentages of parent cells expressing the CD8 marker. (B,C,D) Each point in the bar charts represents the mean of technical triplicates of splenocytes from individual mice (n = 7) as follows: splenocytes from mice immunized with rOprF (black circles), rOprF + SAS (green squares), rOprF + FP18 (red triangles), or rOprF + FP20Rha (blue triangles). Asterisks denote statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Subpopulations of T cells from restimulated splenocytes after immunization with rOprF adjuvanted with FP compounds. (A) Percentages of different populations of CD3⁺CD4⁺ T cells defined by different levels of CD44 and CD62L markers: CD44^low CD62L^hi, naïve; total CD44^hi, activated; CD44^hiCD62L^low, effector memory, and CD44^hi CD62L^hi; central memory. (B) Percentages of gamma-delta T cells and NKT cells. (C) Percentages of natural killer (NK) cells based on their expression of CD49b marker. Asterisks denote statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001. Each point in the graphs indicates the mean of technical triplicates of splenocytes from individual mice (n = 7): splenocytes from mice immunized with rOprF (black circles); rOprF + SAS (green squares); rOprF + FP18 (red triangles); or rOprF + FP20Rha (blue triangles).

Figure 6

Evaluation of the cytokines elicited by CD4⁺ T-cell subtypes in restimulated splenocytes. (A) Total CD4⁺ T cells expressing IFNγ, TNF, IL22, IL17, or IL4 cytokines. (B) Percentages of activated CD4⁺ T cells expressing IFN-γ, TNF, IL22, IL17, or IL4. (C) Percentages of central memory CD4⁺ T cells expressing IFNγ, TNF, IL22, IL17, or IL4. Asterisks denote statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001. Each point in the graphs indicates the mean of technical triplicates of splenocytes from individual mice (n = 7) as follows: splenocytes from mice immunized with rOprF (black circles); rOprF OprF + SAS (green squares); rOprF + FP18 (red triangles); or rOprF + FP20Rha (blue triangles).

Figure 7

Evaluation of the cytokines elicited by CD8⁺ and NK cells in spleens after immunization with FP compounds. (A) Total CD8⁺ T cells expressing IFN-γ, TNF, IL22, or Il4 cytokines. (B) Percentages of NK cells expressing IFN-γ, TNF, IL22 or IL4 cytokines. Asterisks denote statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001. Each point in the graphs indicates the mean of technical triplicates of splenocytes from individual mice (n = 7) as follows: splenocytes from mice immunized with rOprF (black circles); rOprF + SAS (green squares); rOprF + FP18 (red triangles); or rOprF + FP20Rha (blue triangles).